Quantification of the light microscopic analyzes after anti-tyrosine hydroxylase (TH) staining of the olfactory bulb (OB) and the striatal fibers.

<p>No significant differences were seen 2, 5 and 20 days after intranasal application of 0.5(p>0.05, n = 3, striatal fibers were analyzed in a field of 45×45 µm, TH positive neurons were manually counted in the glomerular layer of the OB (total magnification 200×)). Error bars represent SEM.</p

EOG recordings and cookie-finding tests of different mouse strains.

<p>(<b>A</b>) EOG recordings from PaKO and BAsyn mice at 5 months (5 m) and 8 months (8 m) of age. Black and white bars represent the mouse lines listed on the left. n_Parkin = 6, n_BAsyn5m = 5, n_BAsyn8m = 6, n_controls = n_transgenic. Five different dilutions [c] of Henkel 100 were applied (100 ms duration). No significant differences could be detected. (<b>B</b>) Normalized EOG recordings (n = 4) after single odorant applications (geraniol 1∶10, vanillin 40 mM, phenylethylamine (PEA) 1∶1000). Error bars represent SEM. (<b>C</b>) Performances of different mouse lines in the cookie-finding test. Data was normalized to wild type animals and depicts latency to find the cookie. BAsyn mice (8 m) took significantly longer to find the cookie. n_Parkin = 10 (p = 0.75), n_BAsyn = 12 (p = 0.04), n_ThSyn = 10 (p = 0.91), n_controls = n_transgenic. Error bars represent SEM.</p

Olfaction in Three Genetic and Two MPTP-Induced Parkinson’s Disease Mouse Models

<div><p>Various genetic or toxin-induced mouse models are frequently used for investigation of early PD pathology. Although olfactory impairment is known to precede motor symptoms by years, it is not known whether it is caused by impairments in the brain, the olfactory epithelium, or both. In this study, we investigated the olfactory function in three genetic Parkinson’s disease (PD) mouse models and mice treated with MPTP intraperitoneally and intranasally. To investigate olfactory function, we performed electro-olfactogram recordings (EOGs) and an olfactory behavior test (cookie-finding test). We show that neither a parkin knockout mouse strain, nor intraperitoneal MPTP treated animals display any olfactory impairment in EOG recordings and the applied behavior test. We also found no difference in the responses of the olfactory epithelium to odorants in a mouse strain over-expressing doubly mutated α-synuclein, while this mouse strain was not suitable to test olfaction in a cookie-finding test as it displays a mobility impairment. A transgenic mouse expressing mutated α-synuclein in dopaminergic neurons performed equal to control animals in the cookie-finding test. Further we show that intranasal MPTP application can cause functional damage of the olfactory epithelium.</p></div

EOG recordings and cookie-finding tests after intranasal (IN) MPTP application.

<p>(<b>A</b>) Olfactory epithelium after intranasal application of 5 µl blue dye. EOGs were measured in the indicated areas of the OE. (<b>B</b>) EOG data from mice treated wit 0.5 mg/nostril MPTP intranasally. The amplitude was significantly reduced in MPTP treated wildtype (p<0.01), and BAsyn animals (p<0.05), while the rise and the decay time of the responses did not differ (n_MPTP = 6, n_saline = 10). (<b>C</b>) After the training day (day 4), wildtype mice treated IN with MPTP treated took significantly longer to find the cookie 5 days (p<0.01) and 6 days (p<0.05) after MPTP application (n = 10 for each condition), compared to control animals. Finding time of BAsyn mice was not increased after IN MPTP treatment. (<b>D</b>) Animal mobility (mean movement velocity) during the cookie-finding test. Data is normalized to the wild type animals. No significant difference was detectable between saline and MPTP treated animals. BAsyn mice display a significantly reduced mobility (p<0.01). Error bars represent SEM.</p

TH immunostainings of the olfactory bulb and striatum after intranasal MPTP administration.

<p>Light microscopic analysis after anti-tyrosine hydroxylase (TH) staining of the olfactory bulb (OB) and the striatum of BAsyn and wild type mice. Mice were investigated 2, 5 or 20 days (d) after intranasal (IN) MPTP treatment with 0.5 mg/nostril. Scale bars: 50 µm. Pictures are representatives of three biological replicates.</p

Single channel recordings of drPanx1a and drPanx1b.

<p>N2a cells expressing EYFP-tagged drPanx1a or drPanx1b were used for single channel recordings 48 h post transient transfection. (<b>A</b>,<b>B</b>) <i>Single channel recordings of drPanx1 channels from outside-out patches at +30 mV</i>. To elicit channel activity, membrane fragments were processed through depolarizing holding potential steps of (140 s duration; range -60 to +30 mV; increment 10 mV). Both channels display frequent channel activation at positive holding potentials. Arrows in B indicate multiple short-term events. (<b>C</b>,<b>D</b>) <i>Subconductance states of drPanx1 channels from the full 140 s of recording at +30 mV</i>. Intermediate and full open conductance states of the channel are indicated by arrowheads. The full open condition of endogenous mouse Panx1 is labeled by asterisks and was also present in EYFP- and non-transfected controls (data not shown) (c = closed; s = substate; f = fully opened). (<b>E</b>) <i>Comparison of the cumulative full open and closed times of drPanx1 channels</i>. Data were calculated from times for the fully opened and closed state averaged over five total recording periods of 10 s of each membrane fragment at +30 mV. (E left), comparison of the mean times for the fully opened (left) and closed state (right) for five total recording periods of 10 s at +30 mV. (<b>F</b>) Voltage dependent opening probability of drPanx1 channels. (drPanx1a n = 7; drPanx1b n = 10; p<0.05 = *; p<0.01 = **).</p

Analyses of membrane currents elicited by depolarizing voltage ramps in EYFP/Panx1 expressing N2a cells.

<p>N2a cells expressing EYFP or EYFP-tagged mPanx1, drPanx1a or drPanx1b were used for whole-cell patch clamp recordings in the voltage clamp mode 48 h post transient transfection. Current responses to consecutive depolarizing voltage ramps from -60 mV to +80 mV were recorded within the preconditioning paradigm. (<b>A</b>-<b>D</b>) <i>Example traces of the current response elicited by 10 s depolarizing voltage ramps in (A) EYFP, (B) mPanx1, (C) drPanx1 and (D) drPanx1b expressing N2a cells</i>. Asterisks mark the contribution of outward rectifying currents. This causes a decline in the slopes at membrane potentials between +10 mV and +20 mV in all groups, which is barely visible in drPanx1b transfectants. (<b>E</b>) <i>Maximum current amplitudes I_max recorded at +80 mV</i>. (<b>F</b>) <i>Tail </i><i>current </i><i>amplitudes </i><i>I</i>_<i>TC</i><i>evoked </i><i>after </i><i>rapid </i><i>hyperpolarization </i><i>from +80 mV </i><i>to -60mV </i><i>after </i><i>the </i><i>first </i><i>depolarizing </i><i>voltage </i><i>ramp </i><i>and</i> (<b>G</b>) <i>time </i><i>at </i><i>which </i><i>the </i><i>tail </i><i>current </i><i>amplitudes </i><i>decreased </i><i>to 50% of its initial value, T</i>_<i>1/2</i><i>, of </i><i>the </i><i>repolarization </i><i>current</i>. All values in (E-G) were calculated from the averaged current responses to the first voltage ramp within the preconditioning paradigm. Each bar represents the mean + SEM. (EYFP: n = 35; mPanx1: n = 28; drPanx1a: n = 24; drPanx1b: n = 36; p<0.05 = *; p<0.01 = **; p<0.001 = ***).</p

Pannexin1 Channel Proteins in the Zebrafish Retina Have Shared and Unique Properties

<div><p>In mammals, a single pannexin1 gene (Panx1) is widely expressed in the CNS including the inner and outer retinae, forming large-pore voltage-gated membrane channels, which are involved in calcium and ATP signaling. Previously, we discovered that zebrafish lack Panx1 expression in the inner retina, with drPanx1a exclusively expressed in horizontal cells of the outer retina. Here, we characterize a second drPanx1 protein, drPanx1b, generated by whole-genome duplications during teleost evolution. Homology searches strongly support the presence of pannexin sequences in cartilaginous fish and provide evidence that pannexins evolved when urochordata and chordata evolution split. Further, we confirm Panx1 ohnologs being solely present in teleosts. A hallmark of differential expression of drPanx1a and drPanx1b in various zebrafish brain areas is the non-overlapping protein localization of drPanx1a in the outer and drPanx1b in the inner fish retina. A functional comparison of the evolutionary distant fish and mouse Panx1s revealed both, preserved and unique properties. Preserved functions are the capability to form channels opening at resting potential, which are sensitive to known gap junction and hemichannel blockers, intracellular calcium, extracellular ATP and pH changes. However, drPanx1b is unique due to its highly complex glycosylation pattern and distinct electrophysiological gating kinetics. The existence of two Panx1 proteins in zebrafish displaying distinct tissue distribution, protein modification and electrophysiological properties, suggests that both proteins fulfill different functions <i>in vivo</i>.</p> </div

EOG recordings and cookie-finding tests of intraperitoneally (IP) MPTP injected animals.

<p>EOG data from mice injected with MPTP intraperitoneally (<b>A</b>) two days (2d) and (<b>B</b>) five days (5d) after treatment. The amplitudes of the responses, the rise, and the decay times did not differ significantly between the groups. n_IP = 5 animals per condition. (<b>C</b>) Cookie-finding tests after 5 days of IP MPTP injection showed no significant difference between saline and MPTP treated animals (n = 10 per condition). BAsyn animals took significantly longer to find the cookie. (<b>D</b>) Animal mobility (mean movement velocity) during the cookie-finding test. BAsyn animals display a significantly reduced mobility. Error bars represent SEM.</p

Phylogenetic tree of pannexin protein sequences.

<p>Branch colors represent bootstrap values (see right bottom corner). Sequences found in the ghost shark genome are highlighted in red, the sole lancelet sequence found shares similarity with Panx2 sequences (Panx2-like (Panx2l, purple). Zebrafish Panx1a and Panx1b are marked in bold. Tree was rooted to <i>Hydra</i> innexin (Inx) sequences. (ac = A<i>nolis </i><i>carolinensis</i>; bf = <i>Branchiostoma floridae</i>; bt = <i>Bos taurus</i>; ce = <i>Caenorhabditis elegans</i>; cm = <i>Callorhinchus milii</i>; Cx = Connexin; dm = <i>Drosophila melanogaster</i>; dr = <i>Danio rerio</i>; ga = <i>Gasterosteus aculeatus</i>; gg = Gallus gallus; gm = Gadus morhua; hm = <i>Hydra </i><i>magnipapillata</i>; hs = Homo sapiens; lc = <i>Latimeria chalumnae</i>; LRRC = leucine-rich repeat-containing; mg = <i>Meleagris gallopavo</i>; mm = Mus musculus; og = <i>Otolemur garnettii</i>; ol = <i>Oryzias latipes</i>; on = <i>Oreochromis niloticus</i>; Panx = Pannexin; ps = <i>Pelodiscus sinensis</i>; pt = Pan troglodytes; tn = <i>Tetraodon nigroviridis</i>; tr = <i>Takifugu rubripes</i>; tt = <i>Taeniopygia guttata</i>; xt = <i>Xenopus tropicalis</i>).</p